PEITC-mediated inhibition of mRNA translation is associated with both inhibition of mTORC1 and increased eIF2α phosphorylation in established cell lines and primary human leukemia cells.

Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated in vitro with anti-IgM to mimic binding of antigen, a major driver of this leukemia. In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC

ERS statement on standardisation of cardiopulmonary exercise testing in chronic lung diseases

The objective of this document was to standardise published cardiopulmonary exercise testing (CPET) protocols for improved interpretation in clinical settings and multicentre research projects. This document: 1) summarises the protocols and procedures used in published studies focusing on incremental CPET in chronic lung conditions; 2) presents standard incremental protocols for CPET on a stationary cycle ergometer and a treadmill; and 3) provides patients’ perspectives on CPET obtained through an online survey supported by the European Lung Foundation. We systematically reviewed published studies obtained from EMBASE, Medline, Scopus, Web of Science and the Cochrane Library from inception to January 2017. Of 7914 identified studies, 595 studies with 26 523 subjects were included. The literature supports a test protocol with a resting phase lasting at least 3 min, a 3-min unloaded phase, and an 8- to 12-min incremental phase with work rate increased linearly at least every minute, followed by a recovery phase of at least 2–3 min. Patients responding to the survey (n=295) perceived CPET as highly beneficial for their diagnostic assessment and informed the Task Force consensus. Future research should focus on the individualised estimation of optimal work rate increments across different lung diseases, and the collection of robust normative data.The document facilitates standardisation of conducting, reporting and interpreting cardiopulmonary exercise tests in chronic lung diseases for comparison of reference data, multi-centre studies and assessment of interventional efficacy. http://bit.ly/31SXeB

Inhibition of mRNA translation initiation factors as novel therapeutic approach in chronic lymphocytic leukaemia (CLL)

Signalling via the B-cell receptor (BCR) is a major driver of disease progression in chronic lymphocytic leukaemia (CLL) and an established target for therapeutic attack. Studies have demonstrated that BCR-stimulation of CLL cells leads to a substantial increase in global mRNA translation and enhanced translation of oncoprotein MYC. Increased translation is associated with increased expression of eukaryotic initiation factor-4A (eIF4A) in CLL cells, but not in healthy donor B cells, and high expression of eIF4E has been documented in CLL compared to normal B cells. This suggested that it may be possible to selectively inhibit global and/or MYC mRNA translation in CLL cells using inhibitors targeted against specific components of the translation machinery. I therefore investigated the effects of inhibitors of eIF4A, silvestrol and rocaglamide, and an eIF4E inhibitor, ribavirin in CLL.Both eIF4A inhibitors (eIF4Ai) and ribavirin reduced anti-IgM-induced global mRNA translation in primary CLL cells, analysed using O-propargy 1-puromycin (OPP)-labelling. Inhibition of eIF4A resulted in reduced translation of MYC, as well as MCL1, a BCL-2 family protein which, like MYC, is linked to poor outcome. Whilst MYC protein expression was reduced, this was associated with a surprising increase in MYC mRNA expression, via increased RNA stabilisation. Although, eIF4Ai inhibited mRNA translation in healthy donor B cells, inhibition of eIF4E had no effect on translation in B cells from healthy donors. eIF4E also has a role in the nuclear export of specific eIF4E-target mRNAs, including MYC. Ribavirin reduced the nuclear export of mRNA encoding proliferation promoting CCND1 and MYC in CLL samples. In vivo studies utilising ribavirin treatment in mice bearing Eμ-TCL1 leukaemic cells, showed efficacy by reduced tumour burden. Overall, these results support the hypothesis that inhibition of the translation initiation machinery is an effective strategy to suppress anti-IgM-induced translation in CLL cells, to deprive malignant cells of the tumour-promoting effects of oncoproteins such as MYC and MCL1

B-cell receptor signaling induces proteasomal degradation of PDCD4 via MEK1/2 and mTORC1 in malignant B cells

B-cell receptor (BCR) signaling plays a major role in the pathogenesis of B-cell malignancies and is an established target for therapy, including in chronic lymphocytic leukemia cells (CLL), the most common B-cell malignancy. We previously demonstrated that activation of BCR signaling in primary CLL cells downregulated expression of PDCD4, an inhibitor of the translational initiation factor eIF4A and a potential tumor suppressor in lymphoma. Regulation of the PDCD4/eIF4A axis appeared to be important for expression of the MYC oncoprotein as MYC mRNA translation was increased following BCR stimulation and MYC protein induction was repressed by pharmacological inhibition of eIF4A. Here we show that MYC expression is also associated with PDCD4 down-regulation in CLL cells in vivo and characterize the signaling pathways that mediate BCR-induced PDCD4 down-regulation in CLL and lymphoma cells. PDCD4 downregulation was mediated by proteasomal degradation as it was inhibited by proteasome inhibitors in both primary CLL cells and B-lymphoma cell lines. In lymphoma cells, PDCD4 degradation was predominantly dependent on signaling via the AKT pathway. By contrast, in CLL cells, both ERK and AKT pathways contributed to PDCD4 down-regulation and dual inhibition using ibrutinib with either MEK1/2 or mTORC1 inhibition was required to fully reverse PDCD4 down-regulation. Consistent with this, dual inhibition of BTK with MEK1/2 or mTORC1 resulted in the strongest inhibition of BCR-induced MYC expression. This study provides important new insight into the regulation of mRNA translation in B-cell malignancies and a rationale for combinations of kinase inhibitors to target translation control and MYC expression

The dual Syk/JAK inhibitor cerdulatinib antagonizes B-cell receptor and microenvironmental signaling in chronic lymphocytic leukemia

Purpose: B-cell receptor (BCR)-associated kinase inhibitors such as ibrutinib have revolutionised the treatment of chronic lymphocytic leukemia (CLL). However, these agents are not curative and resistance is already emerging in a proportion of patients. Interleukin-4 (IL-4), expressed in CLL lymph nodes, can augment BCR-signalling and reduce the effectiveness of BCR-kinase inhibitors. Therefore simultaneous targeting of the IL-4- and BCR-signalling pathways by cerdulatinib, a novel dual Syk/JAK inhibitor currently in clinical trials (NCT01994382), may improve treatment responses in patients. Experimental Design: PBMCs from CLL patients were treated with cerdulatinib alone or in combination with venetoclax. Cell death, chemokine and cell signalling assay were performed and analysed by flow cytometry, immunoblotting, Q-PCR and ELISA as indicated. Results: at concentrations achievable in patients, cerdulatinib inhibited BCR- and IL-4-induced downstream signalling in CLL cells using multiple read outs and prevented anti-IgM- and nurse-like cell (NLC)-mediated CCL3/CCL4 production. Cerdulatinib induced apoptosis of CLL cells, in a time- and concentration dependent manner, and particularly in IGHV unmutated samples with greater BCR-signalling capacity and response to IL-4, or samples expressing higher levels of sIgM, CD49d+ or ZAP70+. Cerdulatinib overcame anti-IgM, IL-4/CD40L or NLC-mediated protection by preventing upregulation of MCL-1- and BCL-XL, however BCL-2 expression was unaffected. Furthermore in samples treated with IL-4/CD40L, cerdulatinib synergised with venetoclax in vitro to induce greater apoptosis than either drug alone. Conclusion: cerdulatinib is a promising therapeutic for the treatment of CLL either alone or in combination with venetoclax, with the potential to target critical survival pathways in this currently incurable disease<br/

Target-based screening against eIF4A1 reveals the marine natural product elatol as a novel inhibitor of translation initiation with in vivo antitumor activity

Purpose: The DEAD-box RNA helicase eIF4A1 carries out the key enzymatic step of cap-dependent translation initiation and is a well-established target for cancer therapy, but no drug against it has entered evaluation in patients. We identified and characterized a natural compound with broad antitumor activities that emerged from the first target-based screen to identify novel eIF4A1 inhibitors. Experimental Design: We tested potency and specificity of the marine compound elatol versus eIF4A1 ATPase activity. We also assessed eIF4A1 helicase inhibition, binding between the compound and the target including binding site muta-genesis, and extensive mechanistic studies in cells. Finally, we determined maximum tolerated dosing in vivo and assessed activity against xenografted tumors. Results: We found elatol is a specific inhibitor of ATP hydrolysis by eIF4A1 in vitro with broad activity against multiple tumor types. The compound inhibits eIF4A1 helicase activity and binds the target with unexpected 2:1 stoichiometry at key sites in its helicase core. Sensitive tumor cells suffer acute loss of translationally regulated proteins, leading to growth arrest and apoptosis. In contrast to other eIF4A1 inhibitors, elatol induces markers of an integrated stress response, likely an off-target effect, but these effects do not mediate its cytotoxic activities. Elatol is less potent in vitro than the well-studied eIF4A1 inhibitor silvestrol but is tolerated in vivo at approximately 100 relative dosing, leading to significant activity against lymphoma xenografts. Conclusions: Elatol's identification as an eIF4A1 inhibitor with in vivo antitumor activities provides proof of principle for target-based screening against this highly promising target for cancer therapy.</p